Name: intestinal epithelial regeneration in response to ionizing irradiation
Uploaded: 2022-07-27T14:05:00
Description: Watch this Scientific Journal Video about Intestinal Epithelial Regeneration in Response to Ionizing Irradiation at JoVE.com

著者らは、組織標本の準備に関する専門家の支援についてはストーニーブルックがんセンター組織学研究コア、動物の世話と取り扱いを支援するストーニーブルック大学の実験動物資源部門に感謝したいと思います。この研究は、国立衛生研究所からの助成金によって支援されました DK124342 Agnieszka B. Bialkowskaに授与され、DK052230からVincent W. Yang博士に授与されました。

すべてのマウスは、ストーニーブルック大学の実験動物資源部門(DLAR)に収容されました。ストーニーブルック大学施設動物管理および使用委員会(IACUC)は、動物を対象としたすべての研究と手順を承認しました。動物被験者を対象とした実験は、承認された動物取り扱いプロトコル(IACUC #245094)に厳密に従って実施されました。
注:マウス系統B6;129-Bmi1 tm1(cre/ERT)Mrc/J(Bmi...

このプロトコルは、堅牢で再現性のある放射線障害モデルを記述します。これにより、損傷後7日間の腸上皮の変化を正確に分析することができます。重要なことに、選択された時点は損傷の重要な段階を反映しており、腸への明確な変化(損傷、アポトーシス、再生、および正常化段階)によって特徴付けられます60。この照射モデルは確立され、注意深く評価されており、?...

ヒトの腸管上皮は、完全に平らに配置すると、バドミントンコートの表面のほぼ半分を覆います1。代わりに、人間を腸の内容物から分離するこの単一細胞層は、一連の指のような突起、絨毛、およびくぼみ、腸の表面積を最大化する陰窩に圧縮されます。上皮の細胞は陰窩 - 絨毛軸に沿って分化する。絨毛は主に栄養吸収腸細胞、粘液分泌杯細胞、およびホルモン産生腸内分泌細胞で構成され、陰窩は主にディフェンシン産生パネス細胞、活性および予備幹細胞、および前駆細胞2,3,4,5で構成されています。さらに、これらの細胞が下にある間葉系コンパートメントの間質細胞および免疫細胞および内腔の微生物叢との双方向の通信は、腸の恒常性を維持する相互作用の複雑なネットワークを生成し、損傷後の回復に不可欠です6,7,8。
腸上皮は人体で最も急速に自己再生する組織であり、代謝回転率は2〜6日です9,10,11。恒常性の間、ロイシンリッチリピート含有Gタンパク質共役受容体5(LGR5)の発現によって特徴付けられる腸陰窩の基部(陰窩基底柱状細胞)の活性幹細胞は急速に分裂し、他のすべての腸上皮系譜に分化する前駆細胞を提供する。しかし、有糸分裂率が高いため、活性幹細胞とその直接の前駆細胞はガンマ線損傷に特に敏感であり、照射後にアポトーシスを起こします5,12,13,14。それらが失われると、腸陰窩内の予備幹細胞および非幹細胞(前駆細胞の亜集団およびいくつかの最終分化細胞)が活性化され、基底陰窩コンパートメントを補充し、絨毛の細胞集団を再構成し、したがって腸上皮を再生することができる15。系統追跡技術を用いて、複数の研究グループは、予備(静止)幹細胞が活性幹細胞の喪失時の再生をサポートできることを実証している13、16、17、18、19、20、21、22。これらの細胞は、ポリコーム複合体タンパク質1癌遺伝子(Bmi1)、マウステロメラーゼ逆転写酵素遺伝子(mTert)、ホップホメオボックス(Hopx)、およびロイシンリッチリピートタンパク質1遺伝子(Lrig1)の存在によって特徴付けられる。さらに、非幹細胞は、損傷時に腸陰窩を補充することができることが示されている23、24、25、26、27、28、29、30、31。特に、分泌細胞や腸細胞の前駆細胞は、傷害時に脱分化を起こし、幹細胞様細胞に戻り、腸上皮の再生をサポートすることが示されています。最近の研究では、損傷時にステムのような特徴を獲得する能力を持つ複数のマーカー(DLL+、ATOH1+、PROX1+、MIST1+、DCLK1+など)32,33,34,35,36を発現する細胞が同定されています。驚くべきことに、Yuらは、成熟したPaneth細胞(LYZ+)でさえ腸の再生に寄与し得ることを示した37。さらに、腸上皮細胞のアポトーシスを引き起こし、上皮バリア機能を破壊することに加えて、照射は腸内細菌叢の嚥下障害、免疫細胞の活性化および炎症誘発性応答の開始、ならびに間葉系および間質細胞の活性化をもたらす38,39。
ガンマ線は、特に結腸直腸腫瘍の場合、癌治療における貴重な治療ツールです40。しかし、照射は細胞にダメージを与えることで腸の恒常性に大きく影響し、アポトーシスにつながります。放射線被曝は、患者の回復を遅らせる複数の摂動を引き起こし、急性期の粘膜損傷と炎症、および長期的に下痢、失禁、出血、腹痛を特徴としています。この症状の広がりは、胃腸放射線毒性と呼ばれます。さらに、経壁線維症および/または血管硬化症の放射線誘発性の進行は、治療後数年でのみ現れる可能性があります38,41。損傷自体と同時に、放射線は腸細胞の修復応答を誘導し、再生の開始と調整に関与するシグナル伝達経路を活性化します42。放射線誘発性小腸疾患は、他の臓器(子宮頸部、前立腺、膵臓、直腸など)に提供される骨盤または腹部の放射線療法に起因する可能性があります41、43、44、45、46。したがって、腸管照射損傷は重要な臨床的問題であり、結果として生じる病態生理学のより良い理解は、放射線療法に関連する胃腸合併症を軽減するための介入の開発を進める可能性があります。放射線とは別に、腸上皮の再生目的を調査することを可能にする他の技術があります。炎症およびその後の再生を研究するためのトランスジェニックおよびケミカルマウスモデルが開発されている47。デキストラン硫酸ナトリウム(DSS)は腸に炎症を誘発し、炎症性腸疾患と同様の特徴を発達させます48。発がん性化合物アゾキシメタン(AOM)とDSS治療の組み合わせは、大腸炎関連癌の発症をもたらす可能性があります48,49。虚血再灌流誘発傷害は、腸上皮の再生能力を研究するために採用される別の方法である。この技術には経験と外科的知識が必要です50。さらに、前述の技術は放射線とは異なるタイプの傷害を引き起こし、再生の異なるメカニズムの関与につながる可能性があります。さらに、これらのモデルは時間がかかりますが、放射線技術はかなり短いです。最近、腸および結腸から生成されたエンテロイドおよびコロノイドを利用するin vitro法が、腸管再生のメカニズムを研究するために放射線障害と組み合わせて使用 されている51,52。ただし、これらの手法は、モデル53,54の臓器を完全には再現していません。
提示されたプロトコルには、タモキシフェン治療後に予備幹細胞集団に由来する系統の追跡を可能にする遺伝子モデルと組み合わせたガンマ線損傷のマウスモデルの説明が含まれています(Bmi1-CreER;Rosa26eYFP)。このモデルは、12Gy全身照射を利用しており、予備の幹細胞を活性化するのに十分な有意な腸損傷を誘発すると同時に、損傷から7日以内に腸の再生能力のその後の調査を可能にします55。

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			<td>111-545-144</td>
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			<td>11684795910</td>
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			<td>Fisher Scientific</td>
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intestinal epithelial regeneration in response to ionizing irradiation

腸上皮は単層の細胞で構成されていますが、腸管陰窩の底にある腸幹細胞の活発な増殖によって生成される複数の種類の最終分化細胞が含まれています。しかし、急性腸損傷のイベント中に、これらの活性腸幹細胞は細胞死を受けます。ガンマ線照射は広く使用されている結腸直腸癌治療であり、治療的には有効であるが、活性幹細胞プールを枯渇させる副作用がある。実際、患者は放射線療法を受けている間に胃腸放射線症候群を頻繁に経験しますが、これは一部には活発な幹細胞の枯渇によるものです。腸陰窩における活性腸幹細胞の喪失は、典型的には静止している予備腸幹細胞のプールを活性化し、分泌細胞および腸細胞前駆細胞の脱分化を誘導する。これらの細胞がなければ、腸上皮は放射線療法や他のそのような主要な組織傷害から回復する能力を欠いているでしょう。系統追跡技術の新たな進歩により、再生中の細胞の活性化、分化、および移動の追跡が可能になり、腸内でのこれを研究するために首尾よく採用されています。本研究は、放射線障害後のマウス腸管上皮内の細胞を解析する方法を示すことを目的とする。

12 Gy全身照射(TBI)をマウスの遺伝的系統追跡と組み合わせて使用 することで、腸内の放射線損傷の結果を完全に分析することができます。まず、 Bmi1-CreER;Rosa26eYFPマウスは、Bmi1+ 予備幹細胞集団内で黄色蛍光タンパク質(EYFP)発現の増強を誘導するタモキシフェン注射を単回受けた。タモキシフェン注射の2日後に、マウスは照射または偽照射を受けた。安楽死の3?...

Watch this Scientific Journal Video about Intestinal Epithelial Regeneration in Response to Ionizing Irradiation at JoVE.com

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

消化管は、放射線治療によるがん治療の損傷に対して最も敏感な臓器の1つです。それは同時に、そのような侮辱に続く最高の再生能力の1つを持つ臓器系です。提示されたプロトコルは、腸上皮の再生能力を研究するための効率的な方法を記載している。

電離放射線照射による腸管上皮再生

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

ガンマ線照射は、結腸直腸癌の広く使用されている治療法です。そしてそれは治療的に効果的ですが、それは胃腸管に悪影響を及ぼします。提示されたプロトコルは、放射線損傷後の再生中の細胞の活性化、分化、および遊走を研究するための効率的な方法を説明しています。このプロトコルは、堅牢で再現性のある放射線障害モデルを記述します。重要なことに、このモデルは、損傷後の腸細胞の運命を追跡することを可能にし、したがって、特定の細胞亜集団の再生能力のより良い理解を提供する。この手法では、異なる系統追跡動物モデルを使用して、損傷後の細胞のさまざまな亜集団のリアルタイムの行動と相互作用を研究することができます。このプロトコルを少し変更することで、微生物叢と放射線損傷時のさまざまな上皮、間質、および免疫細胞集団との関係を調査できるはずです。はじめに、タモキシフェン粉末を滅菌コーン油にマイクロリットルあたり30マイクログラムの濃度で再懸濁します。60%の振幅で30秒のオンオフサイクルで3分間超音波処理します。室温で1時間暗所で回転させて混合する。次に、滅菌DMSOをEdU粉末に添加してEdUを調製する。全量の1/5を加え、残りの滅菌超純水をゆっくりと加えます。完全に溶解したら、分注して摂氏20度で保存します。水中の70%エタノール、摂氏4度に冷却したDPBS、ブアンの固定液バッファー、10%緩衝ホルマリンなど、組織を採取および固定するための試薬を準備します。次に、マウス解剖キット、ペトリ皿、10ミリリットルの注射器に取り付けられた16ゲージの経管針、組織学的カセットなどの必要な機器を準備します。ガンマ線照射の2日前に、タモキシフェン注射のために実験動物を準備する。各動物の体重を量り、必要な投与量を計算します。腹部を70%エタノールで消毒します。次に、1ミリリットルの注射器に取り付けられた27ゲージの針を使用してタモキシフェンの単回投与を腹腔内に投与し、Creを介した組換えを誘発します。潜在的なタモキシフェン毒性を排除するために、次の48時間動物を観察します。.48時間後、動物を照射室に移します。ガンマ線照射器内のサンプルチャンバーを70%エタノール溶液で消毒します。吸収マットと動物をサンプルチャンバー内に置きます。蓋をして、チャンバーを閉じます。動物を12グレイ全身照射にさらすようにガンマ線照射器をプログラムします。次に、キーを開始位置に回し、開始を押して露出を開始します。アクティブな露出時間のために部屋を出てください。プリセット時間が経過すると、マシンは自動的に停止し、ビープ音が鳴り始めます。キーを停止位置に回して、機械の電源を切ります。サンプルチャンバーを開き、蓋を外します。動物をケージに移し、サンプルチャンバーを70%エタノールで消毒します。動物を従来の飼育室に戻し、処理後の状態を観察します。毎日動物の体重を監視します。計画された安楽死の3時間前に、腹部を消毒し、28ゲージのインスリン注射器を使用して100マイクロリットルのEdUストック溶液をマウスの腹腔内に注射します。動物を安楽死させた後、照射後の異なる時点で近位腸を採取する。小腸の近位部を解剖し、付着した組織を取り除きます。10ミリリットルの注射器に取り付けられた16ゲージのストレート栄養針を使用して、冷たいDPBSで組織を洗い流し、Bouinの固定バッファーで組織を固定します。腸を縦方向に切り開き、スイスロール技術を使用して近位腸を転がします。組織を10%緩衝ホルマリンを含む容器内の組織学的カセットに入れ、室温で24〜48時間インキュベートします。インキュベーション後、組織学的カセットを70%エタノールに移し、組織をパラフィンに包埋します。包埋後、組織学的カセットを少なくとも1時間氷上に置く。次に、ミクロトームを使用して厚さ5ミクロンの水平断面を切り取ります。組織切片を摂氏45度まで温めた水浴に移し、切片を帯電したスライドの上に置きます。スライドを室温で一晩ラックに置いて乾かします。小腸検体のヘマトキシリンおよびエオジン画像は、ゼロ時間で恒常性腸上皮を示し、その後、アポトーシス期に陰窩コンパートメント内の細胞が失われた。これに続いて、高度に増殖する細胞が陰窩に生息する再生段階が続き、7日目までに正常化段階に至りました。EYFPによる系統追跡は、恒常性の間、Bmi1陽性予備幹細胞に由来する細胞が陰窩内のプラス4〜プラス6の位置に制限されることを示した。EdUおよびKi−67染色は、偽照射マウスにおいて正常な腸恒常性を示し、活性幹細胞からトランジット増幅ゾーンを通って拡大した。アポトーシス期には、EYFP陽性細胞の数が減少し、放射線障害によって引き起こされる増殖細胞の喪失を伴いました。再生段階では、予備幹細胞の活性化とBmi1陽性細胞の急速な増殖が観察されました。さらなる増殖および移動は腸上皮の完全性を回復させた。TUNEL染色は、恒常性の間にアポトーシス細胞が存在しないことを示した。TUNEL染色の増加は、アポトーシス期の後半に観察されました。再生段階では、TUNEL染色は再生陰窩内で着実に減少し、陰窩が正常化するとほとんど存在しませんでした。この手法は、RNAシーケンシング、シングルセルRNAシーケンシング、蛍光活性化セルソーティング、またはプロテオミクス解析と組み合わせて、損傷後の腸管再生における特定の細胞系譜の役割に関するより深い知識を集めることができます。研究者らは、腸の再生における微生物叢と上皮、間質、および免疫細胞集団との相互作用を調査して、腸上皮の再生能力の理解を広げることができます。

intestinal-epithelial-regeneration-in-response-to-ionizing-irradiation

Research

JoVE Journal

Biology

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

皮質の神経形成：研究室の進歩からセルベースの​​治療への移行

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

電離放射線は、次のγH2AXの空間分布の評価

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

イモリのレンズ再生を研究するために培養虹彩色素上皮細胞のためのシステム

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

腎上皮再生を研究するためにゼブラフィッシュの前腎のレーザーアブレーション

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

ハイドラ内グルタチオンによって誘発される摂食応答を測定する

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

腸管上皮細胞の透磁率を決定する体外と体内アプローチ

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.